Bacillus anthracis is an important human pathogen that is the etiological agent of anthrax in humans and a potential biological weapon. Two large plasmids, pXO1 and pXO2, play a major role in the virulence of this organism. Gene transfer can frequently occur between B. anthracis and closely related species such as B. cereus and B. thuringiensis, making it likely that the pXO1 and pXO2 plasmids could naturally transfer from B. anthracis into such species that are resistant to antibiotics. Also, the possibility that bioterrorists may introduce the pXO1 and pXO2 plasmids into multiple drug resistant strains to generate "super bioterror agents" cannot be discounted. Although pXO1 is dispensable for the growth of B. anthracis, it is highly stable and rarely lost from growing bacterial cells. Very little is known about the molecular mechanisms involved in the replication and stability of this plasmid. The aims of this proposal are to analyze the replication properties of the anthrax toxin-encoding pXO1 plasmid of B. anthracis. We plan to characterize the minimal replicon of pXO1, including regions involved in plasmid stability and copy number control. We have isolated the replicon of pXO1 and shown that it encodes a novel replication protein, RepX. RepX resembles the FtsZ/tubulin proteins and we have shown that the purified RepX protein is a GTPase that can undergo GTP-dependent dynamic polymerization in vitro. We have also recently shown that RepX polymerizes in vivo in B. anthracis. We hypothesize that RepX may be involved in both the replication and partitioning of the highly stable pXO1 plasmid. Using cell biological approaches with fluorescently labeled proteins and/or pXO1 DNA, we will carry out experiments to study polymerization and localization of RepX in vivo, as well as its co-localization with the pXO1 plasmid and the cell division machinery in vegetatively growing and sporulating B. anthracis cells. By mutational analysis, we will identify regions of RepX that are important for its replication and partitioning activities. Finally, we will utilize "pull-down" assays to identify host- and pXO1-encoded proteins that interact with RepX and may play important roles in pXO1 replication and stability. Our studies could lead to a better understanding of the elements involved in the stable replication and maintenance of the pXO1 plasmid and may contribute to the future development of plasmid-specific co-therapeutic drugs that can reduce the virulence of B. anthracis and related organisms. PUBLIC HEALTH RELEVANCE: The aims of this proposal are to study the role of the RepX protein in the replication and segregation of the anthrax toxin-encoding pXO1 plasmid of Bacillus anthracis. We also plan to study the colocalization of RepX and pXO1 DNA in vivo. We will also investigate the stability and copy number control of pXO1. We plan to identify the functional domains of the RepX protein and identify plasmid- and chromosome-encoded proteins that interact with RepX.